A scientific search for therapeutically useful compounds in combating psychostimulant abuse and addiction has been in progress for decades, with no clinically available agents to date. Rational, brain receptor protein-guided design of novel antidepressants, attention deficit therapeutics and other CNS-acting agents is also an unfulfilled goal of the dopamine transporter (DAT) research field. A major hindrance has been the limited information on the structure and function of the plasma membrane DAT protein. Pharmacological and behavioral studies indicate that the DAT is the brain receptor chiefly responsible for the reward/reinforcing properties of cocaine and the amphetamines. The protein is further implicated in a host of CNS disorders. The long-term objective of this application is to understand how psychostimulant and other CNS-active inhibitors interact with the DAT, which entails mapping DAT ligand binding pockets at the level of the amino acid residue. This level of resolution may now be a possibility, as the recently published crystal structure of the homologous LeuT transporter can serve as a template for creation of 3-D DAT computer models. The specific aims of this proposal entail creation of a LeuT-based 3-D DAT model and use of the model to identify and characterize, via DAT site-directed mutagenesis/pharmacology and crosslinking of novel DAT ligands, the DAT residues key to ligand recognition. The findings will refine the DAT molecular model for in silico screening of structural libraries in the search for novel DAT ligands. These specific aims will be addressed by LeuT and DAT molecular modeling to identify likely ligand binding pocket residues, addressing these targets via site-directed DAT mutagenesis, transfecting mammalian cells with wildtype or mutant DAT nucleic acids, assaying transfected cells for binding and uptake of DAT ligands, assessment of cell surface expression and accessibility of introduced cysteine residues of the mutant transporter proteins via immunoblotting and cysteine alkylation experiments, and immunochemical analysis of DAT proteolysates following covalent attachment of a series of novel DAT ligands. Results from the proposed experiments should reveal specific insights as to how cocaine exert its effects via the DAT, as well as information on DAT recognition of drugs with less abuse potential such as benztropine and methylphenidate. Elucidating at the amino acid residue level the mechanisms of discriminating abused and non-abused substrates and inhibitors may provide a blueprint for rational design of medications that blunt cocaine actions without in turn carrying the potential for abuse and addiction. Additionally, these studies may lead to therapeutics for other DAT-related conditions including depression, anxiety disorders, attention deficit hyperactivity disorder, narcolepsy, chronic pain and Parkinson's disease. PUBLIC HEALTH RELEVANCE: The proposed studies seek to provide 3-dimensional maps illustrating how the dopamine transporter (DAT) interacts with its CNS-active inhibitors;such maps will be used to screen structural databases for novel compounds that function at the DAT. Along the way, map details including how specific DAT protein components contribute to discrimination between, for example, cocaine (high abuse potential) and methylphenidate (low abuse potential) may be elucidated. Findings of this nature open the door to rational design of therapeutics for DAT-related conditions that include substance abuse and addiction, depression, anxiety disorders, attention deficit hyperactivity disorder, narcolepsy, chronic pain and Parkinson's disease.